Download Connections between mRNA 3( end processing and transcription

Connections between mRNA 3( end processing and
transcription termination
Stephen Buratowski
Discoveries within the last few years have revealed that the
multiple steps in gene expression are remarkably integrated.
There have recently been several advances in deciphering how
mRNA 30 end processing is linked with transcription elongation
and termination. It has been known for quite a long time that
transcription termination is somehow intertwined with
polyadenylation, but it is still unclear exactly how these two
processes influence each other. Some recent reports are
shedding light on these connections.
Addresses
Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, 240 Longwood Avenue, Boston,
Massachusetts 02115 USA
Corresponding author: Buratowski, Stephen ([email protected])
Current Opinion in Cell Biology 2005, 17:257–261
This review comes from a themed issue on
Nucleus and gene expression
Edited by Christine Guthrie and Joan Steitz
Available online 12th April 2005
0955-0674/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2005.04.003
Introduction
The connection between polyadenylation and transcription termination was established early on with the discovery that both processes were dependent upon the
same DNA sequences at 30 ends of genes [1,2]. This
connection was further reinforced when it was found that
at least some of the mRNA cleavage and polyadenylation
factors were also required for termination [3–5]. Two
general models were put forward to explain the linkage.
The first, sometimes known as the ‘anti-terminator’
model (Figure 1), proposes that the emergence of the
polyadenylation sequences on the RNA triggers a change
in the factors associated with the polymerase [2]. For
example, binding of the polyadenylation factors could
displace a positive elongation factor or recruit a negative
elongation factor. The consequently less processive RNA
polymerase II (RNApII) would then terminate. In the
second scenario, often called the ‘torpedo’ model
(Figure 2), the cleavage event at the polyadenylation site
generates a new 50 end [1]. Unlike the capped 50 end of
the pre-mRNA, this end could act as an entry point for an
activity (such as an exonuclease or helicase) that would
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track along the RNA and dissociate the polymerase. This
type of mechanism resembles bacterial rho-dependent
termination.
In addition to a requirement for polyadenylation
sequences to trigger termination, several studies have
shown that induced pausing of the polymerase downstream of the polyadenylation site encourages termination [6]. Pausing can be triggered by transcription of
particular DNA/RNA sequences or by a sequence-specific DNA binding protein blocking forward movement of
the RNApII (sometimes referred to as a ‘roadblock’).
Nevertheless, there are clearly no consensus sequences
for the actual site of termination. Instead, termination
apparently occurs stochastically within a window downstream of the polyadenylation site [7,8].
Although there have been many excellent recent reviews
addressing how mRNA processing events are linked to
transcription, this article will concentrate on recent
advances in deciphering how mRNA 30 -end processing
is linked to transcription elongation and termination.
These recent discoveries provide support for both the
‘anti-terminator’ and ‘torpedo’ models.
The C-terminal domain and phosphorylation
Like other mRNA processing events, coupling of 30 end
formation to transcription is mediated by the C-terminal
domain (CTD) of the RNApII largest subunit. This
domain consists of multiple (27–52) repeats of the heptamer sequence YSPTSPS. Serines 2 and 5 of this
sequence are the major sites of CTD phosphorylation.
The current paradigm is that different phosphorylation
patterns predominate at different stages in the transcription cycle and that different proteins bind to specific
phosphorylated forms of the CTD. An oversimplified
incarnation of this model is biphasic, proposing that
the basal transcription factor TFIIH phosphorylates
CTD serine 5 at the promoter and that a second kinase
(Ctk1 in yeast, Cdk9 in higher eukaryotes) phosphorylates CTD serine 2 during elongation phase. However,
the available evidence suggests a more complex and
subtle set of changes. As shown by chromatin immunoprecipitation (ChIP) experiments, serine 2 phosphorylation levels in yeast appear to increase during elongation,
reaching a peak near the polyA site after which they drop
again [9,10,11]. Furthermore, at least some repeats probably remain unphosphorylated and serine 5 phosphorylation levels don’t drop to zero during elongation, indicating
that CTD phosphorylation is not the same at all repeats
[10,12,13]. The Ctk1 kinase can phosphorylate serine 5
Current Opinion in Cell Biology 2005, 17:257–261
258 Nucleus and gene expression
Figure 1
Figure 2
S5P
S2P, S5P
S2P, S5P
pA
P?
pA
RNApII
Poly-A factors
RNApII
Cleavage
Polyadenylation
pA
CTD
RNApII
S5P binding protein (e.g. capping enzyme)
Positive elongation/anti-termination factor
AAAA
Negative elongation/termination factor
A
mRNA release
and export
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The ‘anti-terminator’ model for coupling polyadenylation and
termination. In this model, the extrusion of the polyadenylation signal
(pA) on the RNA results in a change in the factors associated with the
polymerase. Positive elongation factors (light blue oval) may
dissociate or termination factors (purple rectangle) may be recruited.
Also shown are the changing CTD phosphorylation patterns at
different stages of transcription. The larger text size indicates higher
levels of phosphorylation.
pA
RNApII
5′ → 3′ RNA degradation
Termination
RNApII dissociation
Rat1/Xrn2
pA
as well as serine 2 in vitro, and has a significantly higher
activity on serine 2 if serine 5 is phosphorylated first [14].
Therefore, it is likely that at least some repeats of the
CTD are doubly phosphorylated during elongation, adding additional complexity to the potential information
that can be encoded in the CTD [15].
A strong connection has been made between phosphorylation of the CTD by Ctk1/Cdk9 and recruitment of the
polyadenylation factors. First, overexpression of the polyadenylation factor Pti1 suppresses mutant phenotypes
caused by deleting the gene for Ctk1 [16]. Second, the
robust crosslinking of polyadenylation factors normally
seen at 30 ends of genes is no longer observed in strains
lacking Ctk1 [10]. Third, in vivo inhibition of Cdk9 in
Drosophila cells leads to defects in polyadenylation [17].
Finally, in vitro binding of Pcf11 (a subunit of the yeast
polyadenylation factor cleavage factor IA) to the CTD is
dramatically increased by serine 2 phosphorylation [18]. A
co-crystal structure of this interaction shows that the
phosphorylated serine 2 does not make a direct contact
with Pcf11. Instead it may stabilize a particular CTD
conformation that fits the Pcf11 surface [19]. The CTD
interaction domain (CID) of Pcf11 has sequence similarity to two other yeast proteins that function at 30 ends:
Nrd1 [20] and Rtt103 (see below). This suggests that this
particular protein fold serves as a module for targeting to
the CTD of polymerases near the ends of genes.
ChIP experiments show a strong recruitment of polyA
factors and a reduction in CTD phosphorylation just
Current Opinion in Cell Biology 2005, 17:257–261
RNApII
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The ‘torpedo’ model for transcription termination. In this model,
cleavage of the transcript by the polyadenylation machinery (purple
rectangle) generates a new, uncapped 50 -end that is a substrate for
degradation by the Rat1/Xrn2 nuclease. This not only degrades the
‘extra’ part of the mRNA, it also somehow triggers transcription
termination.
downstream of the polyA site. However, the resolution
of this assay doesn’t allow any conclusions to be made
about whether one causes or even precedes the other.
Interestingly, the polyadenylation factor Ssu72 has CTD
phosphatase activity, although it (perhaps unexpectedly)
seems to be specific for phosphorylated serine 5 [21].
Ssu72 also appears to function at promoters [22], raising
several possibilities. Ssu72 may load at the promoter for a
function at 30 ends. Alternatively, Ssu72 phosphatase may
function independently at both 50 and 30 ends. Finally,
Ssu72 may dephosphorylate the CTD at the 30 ends of
genes to facilitate recycling of the polymerase back to a
form competent for initiation. In this respect, a recent
report suggests that the 50 and 30 ends of genes may be
brought into proximity with each by a mechanism that
involves CTD serine 5 phosphorylation [23]. There is
conflicting data as to whether Ssu72 is important for
transcription termination [24–26].
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Connections between mRNA 3( end processing and transcription termination Buratowski 259
Linking termination to polyadenylation
Experimental results in the last few years provide evidence supporting both the ‘anti-terminator’ and the ‘torpedo’ models. It seems unlikely that there is a single
uniform mechanism for RNApII termination. Instead,
there are probably multiple processes that can contribute
to termination, and different processes may predominate
at different genes. Electron microscopy of Drosophila
chromosomes shows different patterns at 30 ends, with
most genes showing abrupt termination without cleaved
transcripts [7,27,28]. This pattern is most consistent with
an ‘anti-terminator’ model. Other genes clearly show
cleaved transcripts associated with elongating polymerases at 30 ends of genes, which is more in line with
the torpedo model.
Cleavage does not seem to be an obligatory precursor to
termination [7,25,27,28]. Studies in mammalian cells
indicate that termination can be separated into two steps:
pausing and polymerase release. Interestingly, the first
step is CTD-independent whereas the second is not [29].
An interesting possibility is that the pausing step reflects a
processivity change in the elongation complex consistent
with the anti-terminator model, while the CTD-dependent release step could be related to recruitment of the
polyadenylation factors by CTD phosphorylation.
A pausing step could be triggered by changing properties
of the elongation complex. Relevant to this, two putative
elongation factors, the PAF and TREX complexes, crosslink throughout the transcribed regions of genes up to the
polyadenylation site. Although the polymerase and several other elongation factors continue further, very little
PAF and TREX crosslinking is seen 30 to the polyA site.
This pattern is independent of CTD phosphorylation by
Ctk1 [10,11]. Interestingly, these two factors, which
were originally found to affect transcription, have also
been implicated in mRNA processing. The TREX complex contains several RNA binding proteins that package
the mRNA for proper splicing and transport [30]. The
function of the PAF complex remains unclear, but recent
experiments suggest that it may also affect polyadenylation [31]. If these two factors act as positive elongation
factors, polymerases downstream of the polyadenylation
site that are not associated with PAF and TREX may be
more competent for termination. Although some crosslinking of polyadenylation factors is sometimes seen in
transcribed regions [18], a very strong signal is seen at and
30 to the polyadenylation site [10,11]. One interesting
possibility is that PAF, TREX or some other RNA binding protein prevents the polyadenylation machinery from
accessing the RNA except when a clear polyA consensus
sequence emerges.
Although it had fallen out of favor in recent years, the
torpedo model of termination has been resurrected by
recent findings. Affinity chromatography with serine 2
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phosphorylated CTD resulted in purification of the
Rtt103 protein, which contains a CID related to that of
the polyA factor Pcf11 [32]. Associated with the RNApII/Rtt103 complex is the Rat1/Rai1 nuclease complex, a
50 -to-30 exonuclease that plays a role in trimming several
ribosomal and small nucleolar RNAs. Rtt103, Rat1, and
Rai1 crosslink at the polyadenylation site, suggesting that
they play a role in 30 end processing. It should be noted,
however, that only Rtt103 crosslinking is dependent upon
CTD phosphorylation by Ctk1. Although cleavage and
polyadenylation are normal in strains mutated for these
factors, a striking defect in termination is seen [32]. The
data strongly support a mechanism in which Rat1 attacks
the new 50 end generated by cleavage. As the polymerase
continues transcribing, the excess transcript is degraded
by Rat1, which eventually reaches the elongation complex and somehow induces termination.
The homolog of yeast Rat1 is in higher eukaryotes is
Xrn2. An RNAi knockdown of Xrn2 also leads to termination defects in mammalian cells [33], suggesting
conservation of an exonuclease-mediated termination
mechanism. One interesting variation on the standard
model for this type of termination has been documented
at the 30 end of the human b-globin gene. In this case, the
RNA downstream of the polyadenylation site can fold
into a self-cleaving ribozyme structure [34]. At this gene,
the Xrn2/Rat1 exonuclease can enter at this co-transcriptional cleavage site rather than the polyA cleavage site. It
remains to be seen whether this observation also applies
to many other genes.
It should be noted that the interval for Rat1 to ‘catch up’
with RNApII could be reduced by the pausing sites
sometimes seen in the 30 regions of genes (see above).
Although it remains unclear whether Rat1 can directly
trigger transcription termination, in vitro studies have
shown that elongation complexes with transcripts <50
nucleotides long are prone to pausing [35]. Contact
between Rat1 and the paused RNApII may lead to
release of the remaining transcript by a mechanism similar to that used by the bacterial rho factor, an RNA
helicase. Alternatively, another enzyme may actually
trigger transcript release. One interesting candidate is
the Sen1 RNA helicase, which has already been implicated in 30 end formation of snoRNAs [20]. Another is
Ttf2/lodestar, a Snf/Swi-family factor that can displace
RNApII from mitotic chromosomes [36].
Conclusions
Given the rapid pace of progress in the area of coupling
between transcription and mRNA processing, it is likely
that the next few years will provide further interesting
surprises. There is still a great deal that remains unknown
about how the polyadenylation factors work, and this will
need to be worked out before we completely understand
how termination is connected. In the meantime, more
Current Opinion in Cell Biology 2005, 17:257–261
260 Nucleus and gene expression
factors connected to other steps in termination will probably be discovered. It will be important to determine
whether these all work together or instead provide multiple independent mechanisms for dissociating transcription elongation complexes.
Acknowledgements
I thank Minkyu Kim for assistance with the figures. This work is
supported by grants GM46498 and GM56663 from the US National
Institutes of Health to SB.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
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showing in vitro binding of polyadenylation factors to the phosphorylated
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Current Opinion in Cell Biology 2005, 17:257–261
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www.sciencedirect.com
Connections between mRNA 3( end processing and transcription termination Buratowski 261
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Buratowski S: The yeast Rat1 exonuclease promotes
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This paper and [33] provide strong support for the torpedo model of
termination. The Rat1/Xrn2 exonuclease is shown to target mRNA 30 ends
and degrade the leftover transcript downstream of the polyadenylation
site. Inactivation of this exonuclease results in read-through of normal
termination sites.
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www.sciencedirect.com
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Here the authors show that termination in the b-globin gene can
be initiated by a self-cleaving ribozyme encoded at the 30 end of
the gene. Like the cleavage generated by the polyA machinery, this
alternative cleavage can generate an entry site for the Rat1/Xrn2
exonuclease.
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Current Opinion in Cell Biology 2005, 17:257–261